Vaccine for periodontal disease

ABSTRACT

The present invention relates to novel bacterial isolates identified by their 16S rRNA DNA, that cause periodontal disease in companion animals, and vaccines comprising such bacteria. Also provided are methods for treating and preventing periodontal disease and kits for detecting and treating periodontal disease kits for detecting and preventing periodontal disease.

FIELD OF THE INVENTION

The present invention relates to novel bacterial isolates identified bytheir 16S rRNA DNA, that cause periodontal disease in companion animals,polynucleotide sequences contained therein, polypeptides encoded by suchpolynucleotide sequences and vaccines comprising such bacterial isolatesthat have been inactivated or attenuated, polynucleotides orpolypeptides. Also provided are methods for treating and preventingperiodontal disease and kits for detecting, treating, and preventingperiodontal disease.

BACKGROUND OF THE INVENTION

Periodontal disease comprises a group of infections involving supportingtissues of the teeth. These range in severity from mild and reversibleinflammation of the gingiva (gum) to chronic destruction ofperiodontal-tissues (gingiva, periodontal ligament, and alveolar bone)with eventual exfoliation of teeth. The vast majority of experimentaldata concerning periodontal diseases is based on studies of humans orbacteria isolated from humans. Relatively little is known with respectto periodontal disease in non-human animals, such as companion animals,and in particular, dogs and cats.

From a microbiological standpoint, several features of this disease areof interest. The bacterial etiology is complex, with a variety oforganisms responsible for the initiation and progression of disease inhumans. Many, if not all, of these organisms may also be present inperiodontally healthy individuals and can exist in commensal harmonywith the host. It is known that in humans, successful colonizers of theteeth and subgingival area must coexist with many (over 600) otherspecies of bacteria that inhabit these regions.

Both the calcified hard tissues of the tooth and the epithelial cells ofthe gingival are available for colonization. These tissues are exposedto host salivary secretions and gingival crevicular fluid (a serumexudate), both of which contain molecules that interact directly withbacteria and alter prevailing environmental conditions. The localenvironment imposes a variety of unique constraints upon the constituentmicrobiota of the supragingival tooth surface and the subgingivalcrevice (the channel between the tooth root and the gingiva that deepensinto a periodontal pocket as disease progresses). Study of thepathogenesis of periodontal diseases in humans is complicated by theecological intricacy of the microenvironment. However, it appears thatdisease episodes may ensue from a shift in the ecological balancebetween bacterial and host factors, as a result of, for example,alteration in the absolute or relative numbers of certain organisms,changes in pathogenic potential, or modulation of particular hostfactors.

The classification of the various manifestations of periodontal diseasein humans is continually changing, and it will suffice to mention thatdiseases range in severity, rate of progression, and number of teethaffected and that different age groups can be susceptible following theeruption of primary teeth. The nature of the pathogenic agents variesamong these disease entities, as well as among human patients and evenbetween different disease sites within a patient. In general, however,severe forms of the disease are associated with a number ofgram-negative anaerobic bacteria. Of this group, in humans, mostevidence points to a pathogenic role for Porphyromonas (formerlyBacteroides) gingivalis. The presence of this organism, acting eitheralone or as a mixed infection with other bacteria, and possibly inconcert with the absence of beneficial species and certain immunologicalresponses in the host, appears to be essential for disease activity.

Initial entry of P. gingivalis into the human oral cavity is thought tooccur by transmission from infected individuals. Other vectors wouldtherefore also appear to be operational. These studies indicate thatindividuals are colonized by a single (or at least a predominant)genotype, regardless of site of colonization or clinical status. Strainsof many different clonal origins, in contrast, are present in differentindividuals. This supports the concept that P. gingivalis is essentiallyan opportunistic pathogen, with virulence not being restricted to aparticular clonal type.

In addition to P. gingivalis, Bacteroides spp. have also been associatedwith periodontitis in man. A novel Bacteroides species, Bacteroidesforsythus, was originally isolated from anaerobic periodontal pockets(Tanner et al., “A study of the bacteria associated with advancingperiodontitis in man”, Journal of Clinical Periodontology (1979), 6,278-307). It was recently reclassified as Tannerella forsythensis basedon various biochemical criteria (Sakamoto et al., “Reclassification ofBacteroides forsythus (Tanner et al. 1986) as Tannerella forsythensiscorrig., gen. nov., comb. nov.”, International Journal of Systematic andEvolutionary Microbiology (2002), 52, 841-849).

While a great deal is known about periodontal disease in humans, verylittle is known about the same disease in companion animals. AlthoughPorphyromonas species have also been implicated in disease in animals,these isolates have characteristics which distinguish them from theirhuman counterparts (reviewed by Harvey in “Periodontal disease in dogs.Etiopathogenesis, prevalence, and significance”, Veterinary Clinics ofNorth America—Small Animal Practice (1998), 28, 1111-1128). Fournier, D.et al. describe the isolation of an animal biotype of P. gingivalis fromvarious animal hosts (“Porphorymonas gulae sp. nov., an Anaerobic,Gram-negative, Coccibacillus from the Gingival Sulcus of Various AnimalHosts”, International Journal of Systematic and EvolutionaryMicrobiology (2001), 51, 1179-1189). The authors hypothesize that thisorganism (P. gulae) represents a Porphyromonas species that is distinctfrom P. gingivalis. WO 03/054755 describes novel Porphyromonas isolatesfrom dogs and cats, as well as methods and kits for treating andpreventing periodontal disease.

Bacteroides species have also been isolated from subgingival sites indogs diagnosed with periodontal disease (Forsblom et al.,“Characterization of Anaerobic, Gram-Negative, Nonpigmented,Saccharolytic Rods from Subgingival Sites in Dogs”, Clinical InfectiousDiseases (1997), 25, S100-106).

There remains a need for a safe and effective vaccine for treating andpreventing periodontal disease in companion animals.

SUMMARY OF THE INVENTION

The invention provides an isolated pigmented anaerobic bacterium whichcauses, either directly or in combination with other pathogenic agents,periodontal disease in companion animals.

In another embodiment, the present invention provides an isolatedpigmented anaerobic bacterium or bacteria which causes, either directlyor in combination with other pathogenic agents, periodontal disease incompanion animals, wherein the bacterium or bacteria can be used toprepare a vaccine for treating or preventing periodontal disease inmammals including companion animals, wherein the vaccine comprises animmunologically effective amount of at least one bacteria or bacteriawhich has/have been inactivated or attenuated.

In one embodiment, the bacterium/bacteria is additionally selected fromthe group consisting of Bacteroides denticanoris, Porphyromonas levii,and Tannerella forsythensis.

Preferably, the bacterium/bacteria comprises a 16S rRNA DNA sequence atleast about 95%, 95.5% 96%, 96.5%, 97%, 97.5% 98%, 98.5%, 99%, 99.5%homologous to a sequence selected from the group consisting of SEQ IDNOS: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15

In yet a further embodiment, the present invention provides an isolatedpolynucleotide molecule comprising any of the nucleotide sequencesselected from the group consisting of SEQ ID NOS: 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14 and 15 and homologues having at least about 95%,95.5% 96%, 96.5%, 97%, 97.5% 98%, 98.5%, 99%, 99.5% homology thereto.The isolated polynucleotides of the invention include fragments andvariants as defined below.

In another aspect, the present invention provides an immunogeniccomposition comprising at least one pigmented anaerobic bacteriaaccording to the present invention, and a pharmaceutically acceptablecarrier. The bacteria of the immunogenic composition may be live orinactivated. Optionally the immunogenic composition may include anadjuvant.

In a further aspect, the present invention provides a vaccine fortreating or preventing periodontal disease in mammals includingcompanion animals comprising an immunologically effective amount of atleast one pigmented anaerobic bacteria according to the presentinvention, and a pharmaceutically acceptable carrier. The bacteria ofthe vaccine may be live or inactivated. Optionally the vaccine mayinclude an adjuvant.

In another aspect the present invention provides a method for treatingor preventing periodontal disease in mammals including companion animalscomprising administering to a mammal in need thereof, a vaccinecomposition according to the present invention.

In another aspect the present invention provides a method for diagnosingperiodontal disease in mammals including companion animals by analyzinga sample for bacteria, polypeptides or polynucleotides of the presentinvention, wherein the presence of the bacteria, polypeptides, orpolynucleotides are indicative of disease. Preferably, the analyzingstep includes analyzing the sample using a method selected from thegroup consisting of PCR, hybridization, and antibody detection.

In yet another aspect, the present invention provides a kit comprising,in at least one container, a composition for treating and preventingperiodontal disease in mammals including companion animals comprising aneffective amount of at least one Inactivated or attenuated isolatedpigmented anaerobic bacteria, or a polypeptide, or polynucleotidesderived from the pigmented anaerobic bacteria and a pharmaceuticallyacceptable carrier; The kit further comprises a set of printedinstructions indicating that the kit is useful for treating orpreventing periodontal disease in mammals. The kit may further comprisea means for dispensing said composition.

In still another aspect, the present invention provides a kit comprisingin at least one container an isolated DNA molecule comprising anucleotide sequence of at least about 15 contiguous nucleotides selectedfrom any of SEQ ID NOS: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15which hybridizes under highly stringent conditions to the complement ofany of the nucleotide sequences depicted in SEQ ID NOS: 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13,14 and 15 and a second isolated DNA moleculecomprising in a second container an isolated DNA molecule comprising anucleotide sequence of at least about 15 contiguous nucleotides selectedfrom the complement of any of the nucleotide sequences depicted in SEQID NOS: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 which hybridizesunder highly stringent conditions to any of the nucleotide sequencesdepicted in SEQ ID NOS: 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14 and 15wherein the kit further comprises a set of instructions indicating thatthe kit is useful for the detection of Bacteroides, Porphyromonas, andTannerella spp. Such a method may be used generally in all mammalsincluding companion animals.

In a further aspect, the present invention provides a hybridization kitcomprising in at least one container an isolated DNA molecule comprisinga nucleotide sequence of at least about 15 contiguous nucleotidesselected from any of SEQ ID NOS: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14and 15 or its complement, wherein the hybridization is specific toBacteroides, Porphyromonas, and Tannerella spp. and wherein the kitfurther comprises a set of instructions indicating that the kit Isuseful for the detection of Bacteroides, Porphyromonas, and Tannerellaspp. Preferably, the hybridization is performed under highly stringentconditions.

The invention further provides a biologically pure culture of bacteria,wherein the bacteria comprise a 16S rRNA DNA sequence at least about95%, 95.5% 96%, 96.5%, 97%, 97.5% 98%, 98.5%, 99%, 99.5% homologous to asequence selected from the group consisting of SEQ ID NOS: 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 and 15.

The invention also provides a biologically pure culture of bacteriawhich is ATCC PTA-5881 or a culture having all of the identifyingcharacteristics of ATCC PTA-5881. The invention also provides abiologically pure culture of bacteria which is ATCC PTA-5882 or aculture having all of the identifying characteristics of ATCC PTA-5882.The invention provides a biologically pure culture of bacteria which isATCC PTA-6063 or a culture having all of the identifying characteristicsof ATCC PTA-6063.

The invention also comprises isolated polynucleotides and polypeptidesderived from the bacteria of the invention which have utility as avaccine for treating or preventing periodontal disease in mammalsincluding companion animals.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Canine and feline BPAB isolate characterization

FIG. 2. The results of RapID ANA II testing for B. denticanoris B78^(T)as well as six control bacteria.

FIG. 3. Neighbor-joining phylogenetic tree for representatives from theBacteroidetes class. The phylogenetic tree was generated using theCLUSTAL X version 1.81 and NJ Plot software programs (both availablefrom ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/). The tree was rooted tothe Escherichia coli 16S rRNA gene sequence (accession number J01695)(data not shown). Bootstrap analysis was performed using 1000replicates. Bootstrap values are presented graphically (•>950; ▪, >850;o, >700; □, >500; no designation, <500). The scale bar represents 0.01substitutions per nucleotide position. The arrow indicates the locationof B. denticanoris B78^(T). Accession numbers: P. gingivalis ATCC 33277,J01695; P. gulae B243, AF285874; P. cansulci VPB 4875, X76260; P.salivosa NCTC 11632, L26103; P. endodontalis ATCC 35406, AY253728; T.forsythensis ATCC 43037, AB035460; Bacteroides cf. forsythus oral cloneBU45, AF385565; B. merdae ATCC 43184T, X83954; B. distasonis ATCC 8503,M86695; Equine fecal bacterium 118ds10, AY212569; D. shahii strain CCUG43457, AJ319867; A. putredinis ATCC 29800, L16497; R. microfusus ATCC29728, L16498; Swine fecal bacterium FPC111, AF445205; Bacteroides sp.139, AF319778; B. fragilis ATCC 25285T, X83935; B. thetaiotaomicronstrain 17.4, AY319392; B. acidofaciens strain A37, AB021163; B.denticanoris B7₈ ^(T), AY549431; Bacteroides sp. 0103-800, AJ416906;Uncultured Bacteroidetes Bisii27, UBA318179; P. bivia ATCC 29303.L16475; P. nigrescens ATCC 25261, L16479; P. intermedia ATCC 25611,L16468; P. denticola ATCC 35308, L16467; and P. buccae ATCC 33690,L16478.

FIG. 4. Neighbor-joining phylogenetic tree for clinical isolates of B.denticanoris. The phylogenetic tree was generated as in FIG. 1. The treewas rooted to the Bacteroides sp. 0103-800 16S rRNA gene sequence(accession number AJ416906). The scale bar represents 0.001substitutions per nucleotide position. Only one member from each 16SrRNA sequence cluster is shown. Accession numbers: B. denticanoris B7₈^(T), AY549431; B. denticanoris B80, AY549432; B. denticanoris B83,AY549433; B. denticanoris B241, AY549434; B. denticanoris B242,AY549435; B. denticanoris B342, AY549436; B. denticanoris B458,AY549437; B. denticanoris B473, AY549438; B. denticanoris B474,AY549439; and B. denticanoris B476, AY549440.

FIG. 5. Pathogenicity testing of B. denticanoris B78^(T) in the oralmouse model of periodontal disease. Sixteen mice were used for each testgroup. Mice were treated as described. Forty-two days post challenge,the mice were sacrificed, the jaws defleshed and stained. Fourteenindependent measurements of the CEJ-ABC distance were taken on each jaw.The average CEJ-ABC measurement for each group is shown. The standarderror for each group is indicated. The statistical significance betweenthe two groups is shown.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

-   Seq ID No. 1—Sequencing Primer-   Seq ID No. 2—Sequencing Primer-   Seq ID No. 3—DNA encoding a portion of the 16S rRNA from Bacteroides    denticanoris (B78)-   Seq ID No. 4—DNA encoding a portion of the 16S rRNA from    Porphyromonas levii (B222)-   Seq ID No. 5—DNA encoding a portion of the 16S rRNA from Tannerella    forsythensis (B343-24)-   Seq ID No. 6—DNA encoding a portion of the 16S rRNA from Bacteroides    denticanoris (B78) (full length)-   Seq ID No. 7—DNA encoding a portion of the 16S rRNA from Bacteroides    denticanoris (B80)-   Seq ID No. 8—DNA encoding a portion of the 16S rRNA from Bacteroides    denticanoris (B83)-   Seq ID No. 9—DNA encoding a portion of the 16S rRNA from Bacteroides    denticanoris (B241)-   Seq ID No. 10—DNA encoding a portion of the 16S rRNA from    Bacteroides denticanoris (B242)-   Seq ID No. 11—DNA encoding a portion of the 16S rRNA from    Bacteroides denticanoris (B342)-   Seq ID No. 12—DNA encoding a portion of the 16S rRNA from    Bacteroides denticanoris (B458)-   Seq ID No. 13—DNA encoding a portion of the 16S rRNA from    Bacteroides denticanoris (B473)-   Seq ID No. 14—DNA encoding a portion of the 16S rRNA from    Bacteroides denticanoris (B474)-   Seq ID No. 15—DNA encoding a portion of the 16S rRNA from    Bacteroides denticanoris (B476)-   Seq ID No. 16—Sequencing Primer-   Seq ID No. 17—Sequencing Primer

DETAILED DESCRIPTION OF THE INVENTION Bacterial Isolates

The present invention provides isolated anaerobic bacteria, identifiedby their 16S rRNA DNA sequences, which can cause periodontal disease andvarious other diseases and clinical manifestations in companion animals.More specifically, the bacteria are selected from the generaBacteroides, Porphyromonas, and Tannerella.

In addition the invention provides a novel, anaerobic bacteria/bacteriumcausing periodontal disease in companion animals. The novel isolateinduces alveolar bone loss in a mouse model of experimental periodontaldisease The cellular morphology and biochemical properties of thebacterial isolate indicates that it is a member of the genusBacteroides. Comparison of the 16S rRNA gene sequence suggested that thebacteria represented a previously undefined species within the genusBacteroides based on biochemical, molecular phylogenetic, and pathogenicevidence, which we have designated Bacteroides denticanoris sp. nov. Thetype strain of Bacteroides denticanoris is strain B78^(T) (=ATCCPTA-5881). Preferably, therefore the isolated bacteria of the presentinvention include Bacteroides denticanoris (B78), Porphyromonas levii(B222), and Tannerella forsythensis (B343-24), although other species orstrains are encompassed by the invention. In a preferred embodiment, theisolated bacteria of the present invention can be identified by their16S rRNA DNA sequences shown in SEQ ID Nos. 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 and 15.

The diseases caused by infection with the bacteria of the presentinvention include, but are not limited to, companion animal periodontaldisease, companion animal oral malodor (halitosis), bovine foot rot,canine coronary heart disease and canine systemic infections. Bacteriawithin these genera have also been connected with various humandiseases, including coronary heart disease, parotitis, oral malodor,gingivitis, periodontis, stroke, atherosclerosis, hyperlipidemia,bacterial vaginosis, intrauterine growth retardation (IUGR), andincreased incidence of pre-term delivery of low birth weight infants.

The present invention provides isolated polynucleotide molecules ofbacterial species. The present invention also provides polynucleotidesequences having at least about 90% homology, preferably at least about95%, 95.5%. 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, sequence identityto any of SEQ 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15.

In addition, the present invention provides polynucleotide sequencesthat hybridize under stringent conditions to the complement of any ofthe polynucleotide sequences shown in SEQ ID 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14 and 15.

In another specific embodiment, a nucleic acid which is hybridizable toany of the polynucleotide sequences depicted in SEQ ID 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14 and 15, or their complements, under conditions ofhigh stringency is provided. By way of example and not limitation,procedures using such conditions of high stringency for regions ofhybridization of over 90 nucleotides are as follows. Prehybridization offilters containing DNA is carried out for 8 h to overnight at 65° C. inbuffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP,0.02% Ficoll, 0.02% BSA, and 500 μg/mL denatured salmon sperm DNA.Filters are hybridized for 48 h at 65° C. in prehybridization mixturecontaining 100 μg/mL denatured salmon sperm DNA and 5-20×10⁶ cpm of³²P-labeled probe. Washing of filters is done at 37° C. for 1 h in asolution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. Thisis followed by a wash in 0.1×SSC at 50° C. for 45 min beforeautoradiography.

Other conditions of high stringency which may be used depend on thenature of the nucleic acid (e.g. length, GC content, etc.) and thepurpose of the hybridization (detection, amplification, etc.) and arewell known in the art. For example, stringent hybridization of anoligonucleotide of approximately 15-40 bases to a complementary sequencein the polymerase chain reaction (PCR) is done under the followingconditions: a salt concentration of 50 mM KCl, a buffer concentration of10 mM Tris-HCl, a Mg²⁺ concentration of 1.5 mM, a pH of 7-7.5 and anannealing temperature of 55-60° C.

In a preferred embodiment, after hybridization, wash conditions are asfollows. Each membrane is washed two times each for 30 minutes each at45° C. in 40 mM sodium phosphate, pH 7.2, 5% SDS, 1 mM EDTA, 0.5% bovineserum albumin, followed by four washes each for 30 minutes in sodiumphosphate, pH 7.2, 1% SDS, 1 mM EDTA. For high stringency hybridization,the membranes are additionally subjected to four washes each for 30minutes in 40 mM sodium phosphate, pH 7.2, 1% SDS, 1 mM EDTA at 55° C.,followed by four washes each for 30 minutes in sodium phosphate, pH 7.2,1% SDS, 1 mM EDTA at 65° C.

The present invention further provides vaccines and vaccine formulationswhich, when administered to a companion animal in a therapeuticallyeffective amount, are useful in treating or preventing (i.e., conferringresistance) to periodontal disease in a companion animal.

In one embodiment, the present invention provides a vaccine thatcomprises at least one attenuated (modified live) or inactivated wholecell preparation (bacterin). In another embodiment, the vaccinecomprises a subunit fraction from one or more bacterial species,capableof inducing an immune response.

The attenuated (modified live) or inactivated vaccines (bacterins) canbe present in combination with other known vaccine formulationcomponents such as with compatible adjuvants, diluents, or carriers.

DEFINITIONS AND ABBREVIATIONS

The term “identity” or “percentage of sequence identity” for nucleotidesequences is determined by comparing two optimally aligned sequencesover a comparison window, wherein optimal alignment provides the highestorder match and can introduce nucleotide additions or to the test orreference sequence. The percentage identity is determined by calculatingthe percentage of nucleotides that are identical between the test andreference sequence at each position over the entire sequence. Optimalsequence alignment and percentage identity can be determined manually,or more preferably by a computer algorithm including but not limited toTBLASTN, FASTA, GAP, BESTFIT, and CLUSTALW (Altschul et al., 1990, J.Mol. Biol. 215(3):403-10; Pearson and Lipman, 1988, Proc. Natl. Acad.Sci. USA 85(8):2444-8; Thompson, et al., 1994, Nucleic Acids Res.22(22):4673-80; Devereux et al., 1984, Nuc. Acids. Res. 12:387-395;Higgins, et al., 1996, Methods Enzymol. 266:383-402). Preferably, theNCBI Blast Server (http://www.ncbi.nlm.nih.gov) set at the defaultparameters is used to search multiple databases for homologoussequences.

The term “heterologous”, when used herein means derived from a differentbacterial species or strain.

The term “homology”, “homologous”, and the like, when used herein meansthe degree of identity shared between polynucleotide or polypeptidesequences.

The term “homologous”, when used in reference to a bacterial speciesmeans the same bacterial species or strain.

The term “isolated” when used herein means removed from its naturallyoccurring environment, either alone or in a heterologous host cell, orchromosome or vector (e.g., plasmid, phage, etc.).

The terms “isolated anaerobic bacteria”, “isolated bacteria”, “isolatedbacterial strain” and the like refer to a composition in which thebacteria are substantially free of other microorganisms, e.g., in aculture, such as when separated from it naturally occurring environment.The term “biologically pure culture” when applied to the bacteria of theinvention refers to a culture of bacteria substantially free of othermicroorganisms.

The term “isolated polynucleotide” indicates a composition in which theisolated nucleotide comprises at least 50% of the composition by weight.More preferably, the isolated polynucleotide comprises about 95%, andmost preferably 99% by weight of the composition.

The term “functionally equivalent” as utilized herein, refers to arecombinant polypeptide capable of being recognized by an antibodyspecific to native polypeptide produced by the bacteria which causesperiodontal disease in companion animals, or a recombinant polypeptidecapable of eliciting or causing a substantially similar immunologicalresponse as that of the native protein from the endogenous bacteria.Thus, an antibody raised against a functionally equivalent polypeptidealso recognizes the native polypeptide produced by the bacteria whichcauses periodontal disease in companion animals.

The term “immunogenicity” refers to the capability of a protein orpolypeptide to elicit an immune response directed specifically againstthe bacteria that causes periodontal disease in companion animals.

The term “antigenicity” refers to the capability of a protein orpolypeptide to be immunospecifically bound by an antibody raised againstthe protein or polypeptide.

The term “antibody”, as used herein, refers to an immunoglobulinmolecule able to bind to an antigen. Antibodies can be a polyclonalmixture or monoclonal. Antibodies can be intact immunoglobulins derivedfrom natural sources or from recombinant sources, or can beimmunoreactive portions of intact immunoglobulins. Antibodies can existin a variety of forms including, for example, as, Fv, Fab′, F(ab′)₂, aswell as in single chains.

The term “companion animal”, as used herein, refers to any non-humananimal in captivity considered to be a pet. These may include, but arenot restricted to, dogs, cats, horses, rabbits, monkeys, and rodents,including mice, rats, hamsters, gerbils, and ferrets.

The term “protection”, “protecting”, and the like, as used herein withrespect to a vaccine, means that the vaccine prevents or reduces thesymptoms of the disease caused by the organism from which the antigen(s)used in the vaccine is derived. The terms “protection” and “protecting”and the like, also mean that the vaccine can be used to “treat” thedisease or one of more symptoms of the disease that already exists in asubject.

The term “therapeutically effective amount” refers to an amount of thebacteria, or a subunit, (e.g., polypeptides, polynucleotide sequences)and combinations thereof sufficient to elicit an immune response in thesubject to which it is administered. The immune response can comprise,without limitation, induction of cellular and/or humoral immunity.

The term “preventing infection” means to prevent or inhibit thereplication of the bacteria which cause periodontal disease in companionanimals, to inhibit transmission of the bacteria, or to prevent thebacteria from establishing itself in its host, or to alleviate thesymptoms of the disease caused by infection. The treatment is consideredtherapeutic if there is a reduction in bacterial load.

The term “pharmaceutically acceptable carrier” refers to a carriermedium that does not interfere with the effectiveness of the biologicalactivity of the active ingredient and is not toxic to the subject towhom it is administered.

The term “therapeutic agent” refers to any molecule, compound ortreatment, preferably an antibacterial, that assists in the treatment ofa bacterial infection or a disease or condition caused thereby.

The term “fragment or variant thereof” refers to partial nucleotidesequences according to the present invention. Analogs are encompassed bythe term “fragment or variant thereof”. Mutant polynucleotides which maypossess one or more mutations which are deletions, insertions orsubstitutions of nucleotide residues are encompassed by the term“fragment or variant thereof”. Allelic variants are encompassed by theterm “fragment or variant thereof”.

Isolation and Characterization of Bacterial Species

Bacteria provided by the present invention can be obtained using knownsampling, culture and isolation techniques. For example, microbialsamples can be obtained from a population of companion animals, such asfrom dogs and cats, exhibiting periodontal disease. Evidence ofperiodontal disease can be observed using known measures, such as dogswith periodontal pockets >3 mm and cats with periodontal pockets >2 mm.Known parameters for characterizing periodontal disease such as dentalindices (gingival index and periodontal index) and periodontal pocketdepths can determined for the sample population of companion animals.Individual samples can be obtained from the periodontal pocket of aparticular animal, maintained under anaerobic conditions and culturedusing various known culture media.

Clinical isolates can be characterized using known techniques such as anumber of biochemical tests, and 16S rRNA DNA sequence analysis todetermine their genus and species. Individual isolates can betransferred to plates and antibiotic disks (Anaerobe Systems) can beplaced on the agar surface to determine the antibiotic resistancepatterns of each isolate. Purified colonies can also be subjected toknown indole and catalase tests (Anaerobe Systems). Lipase andlecithinase production patterns can be determined for individualisolates.

The isolates can be typed based on their 16S rRNA DNA sequence.Individual, well-isolated colonies can be utilized as a template forpolymerase chain reactions (PCR) amplification of the 16S rRNA regionusing, for example, primers D0056 and D0057 (Seq. ID NO. 1 and Seq. IDNO. 2; Table 1). Optionally the full length 16S RNA can be amplifiedusing, for example, the primers disclosed as Seq ID NO. 16 and 17.)

The resulting PCR products can be purified using available PCR prepskits (Promega Corp.; Madison, Wisc.) and pooled by isolate. The purifiedPCR products can then be desalted and subjected to DNA sequenceanalysis. The resulting DNA sequences can be used to search availableDNA databases. The bacterial isolates can then be typed based on theclosest match identified by database searches. TABLE 1 DNA sequenceidentification listing. All oligonucleotide primers were synthesized byGibco-BRL (USA). SEQ ID NO. Name Target DNA Sequence 1 D0056 16S rRNAGGATTAGATACCCTGGTAGTC 2 D0057 16S rRNA CCCGGGAACGTATTCACCG 3 Bacteroides(Not GCACAGTAAACGATGAATACTCGCTGTTT denticanoris applicable)GCGATACACTGTAAGCGGCCAAGCGAAA (B78) 16S rRNA GCGTTAAGTATTCCACCTGGGGApolynucleotide GTACGCCGGCAACGGTGAAACTCAAAGG sequenceAATTGACGGGGGCCCGCACAAGCGGAG GAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGGGCTTAAATT GCGCTGGCTTTTACCGGAAACGGTATTTTCTTCGGACCAGCGTGAAGGTGCT GCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAAGTGCCATAACGAGCG CAACCCTTATCTTTAGTTACTAACAGTTTTGCTGAGGACTCTAAAGAGACTG CCGTCGTAAGATGCGAGGAAGGTGGGGATGACGTCAAATCAGCACGGCCCTT ACGTCCGGGGCTACACACGTGTTACAATGGGGAGCACAGCAGGTTGCTACACGGC GACGTGATGCCAATCCGTAAAACTCCTCTCAGTTCGGATCGAAGTCTGCAACC CGACTTCGTGAAGCTGGATTCGCTAGTA ATCGCGCATCAGCC4 Porphyromonas (Not CGCTGTAAACGATGATTACTCAGAGTATG levii (B222)applicable) CGATATAATGTATGCTCTCAAGCGAAAGC 16S rRNAGTTAAGTAATCCACCTGGGGAG polynucleotide TACGTCGGCAACGATGAAACTCAAAGGAsequence ATTGACGGGGGCCCGCACAAGCGGAGG AACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCTGGGATTGAAATG TATATGCCGGTATCCCGAAAGGGGTGCTATTCACTTCGGTGACGTATATGTA GGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAAGTGCCATAA CGAGCGCAACCCTTATCGTCAGTTGCTAGCAGGTAAAGCTGAGGACTCTGGC GAGACTGCCGTCGTAAGGCGAGAGGAAGGTGGGGATGACGTCAAATCAGCAC GGCCCTTATATCCAGGGCGACACACGTGTTACAATGGTGAGGACAAAGGGTCGCTA CCCGGTGACGGGATGCCAATCTCCAAACCTCATCTCAGTTCGGATCGGAGTC TGCAACTCGACTCCGTGAAGCTGGATTCGCTAGTAATCGCGCATCAGCCATG 5 Tannerella (Not TACTAGGAGTTTGCGATATACAGTAAGCTforsythensis applicable) CTACAGCGAAAGCGTTAAGTAATCCACC (B343-24) 16STGGGGAGTACGCCGGCAACGGTG rRNA AAACTCAAAGGAATTGACGGGGGCCCGC polynucleotideACAAGCGGAGGAACATGTGGTTTAATTC sequence GATGATACGCGAGGAACCTTACCCGGGATTGAAATGTAGACGACGGACAGTG AGAGCTGTCTTCCCTTCGGGGCGTCTATGTAGGTGCTGCATGGTTGTCGTCA GCTCGTGCCGTGAGGTGTCGGCTTAAGTGCCATAACGAGCGCAACCCTGACTGTCA GTTGCTAACAGGTTAAGCTGAGGACTCTGGCGGGACTGCCGGCGTAAGCTG TGAGGAAGGTTGGGATGACGTCAAATCAGCACGGCCCTTACATCCGGGGCGAC ACACGTGTTACAATGGCAGGGACAAAGGGCAGCTACCGGGCGACCGGATGCCAAT CTCCAAACCCTGTCTCAGTTCGGATCGGAGTCTGCAACTCGACTCCGTGAAGC TGGATTCGCTAG

The following companion animal periodontal isolates were deposited withthe American Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Va., 20110, USA: Bacteroides denticanoris (B78; (PTA-5881),Porphyromonas levii (B222; (PTA-5882), and Tannerella forsythensis(B343-24; (PTA-6063).

Cloning of Bacterial Nucleotide Sequences

There are several known methods or techniques that can be used to clonethe nucleotide sequences of the present invention. For example, thesequences can be isolated as restriction fragments and cloned intocloning and/or expression vectors, the sequences can be PCR amplifiedand cloned into cloning and/or expression vectors, or the sequences canbe cloned by a combination of these two methods.

Standard molecular biology techniques known in the art and notspecifically described can be generally followed as described inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York (1989); Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1989); Perbal, A Practical Guide to Molecular Cloning, John Wiley &Sons, New York (1988); Watson et al., Recombinant DNA, ScientificAmerican Books, New York; Birren et al (eds) Genome Analysis: ALaboratory Manual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press,New York (1998); and methodology set forth in U.S. Pat. Nos. 4,666,828;4,683,202; 4,801,531; 5,192,659 and 5,272,057. Polymerase chain reaction(PCR) is carried out generally as described in PCR Protocols: A Guide ToMethods And Applications, Academic Press, San Diego, Calif. (1990).

Examples of methods useful in cloning and sequencing the polynucleotidesof the present invention are provided in the Example.

Antibody Production

Antibodies may either be monoclonal, polyclonal, or recombinant.Conveniently, the antibodies may be prepared against the immunogen orportion thereof, or prepared recombinantly by cloning techniques or thenatural gene product and/or portions thereof may be isolated and used asthe immunogen. Immunogens can be used to produce antibodies by standardantibody production technology well known to those skilled in the art asdescribed generally in Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988 andBorrebaeck, Antibody Engineering—A Practical Guide, W.H. Freeman andCo., 1992. Antibody fragments may also be prepared from the antibodiesand include Fab, F(ab′)₂, and Fv by methods known to those skilled inthe art.

In the production of antibodies, screening for the desired antibody canbe accomplished by standard methods in immunology known in the art.Techniques not specifically described are generally followed as inStites et al.(eds), Basic and Clinical Immunology (8th Edition),Appleton & Lange, Norwalk, Conn. (1994) and Mishell and Shiigi (eds),Selected Methods in Cellular Immunology, W.H. Freeman and Co., New York(1980). In general, ELISAs and Western blotting are the preferred typesof immunoassays. Both assays are well known to those skilled in the art.Both polyclonal and monoclonal antibodies can be used in the assays. Theantibody can be bound to a solid support substrate or conjugated with adetectable moiety or be both bound and conjugated as is well known inthe art (for a general discussion of conjugation of fluorescent orenzymatic moieties see Johnstone & Thorpe, Immunochemistry in Practice,Blackwell Scientific Publications, Oxford, 1982.) The binding ofantibodies to a solid support substrate is also well known in the art(see for a general discussion, Harlow & Lane Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Publications, New York, 1988 andBorrebaeck, Antibody Engineering—A Practical Guide, W.H. Freeman andCo., 1992). The detectable moieties contemplated for use in the presentinvention can include, but are not limited to, fluorescent, metallic,enzymatic and radioactive markers such as biotin, gold, ferritin,alkaline phosphatase, b-galactosidase, peroxidase, urease, fluorescein,rhodamine, tritium, ¹⁴C and iodination.

Where appropriate, other immunoassays such as radioimmunoassays (RIA)can be used as known in the art. Available immunoassays are extensivelydescribed in the patent and scientific literature. See, for example,U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987;3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345;4,034,074; 4,098,876; 4,879,219; 5,011,771; and 5,281,521, as well asSambrook et al, Molecular Cloning: A Laboratory Manual, Cold SpringsHarbor, N.Y., 1989.

Detection, Diagnostic, and Prevention Kits

The present invention further provides kits for the detection ofBacteroides, Porphyromonas, and Tannerella species. The kit includesreagents for analyzing a sample for the presence of said organisms,polypeptides, or nucleotide sequences of the present invention, whereinthe presence of the nucleotide sequence is indicative of the presence ofthe organism. This method is valuable because disease can be diagnosedprior to the existence of symptoms and can therefore prevent the onsetof the disease prior to the occurrence of damage to the patient. Thepresence of bacteria, polypeptides or nucleotide sequences can bedetermined using antibodies, PCR, hybridization, and other detectionmethods known to those of skill in the art.

In one embodiment, the kit provides reagents for the detection ofantibodies against Bacteroides, Porphyromonas, or Tannerella spp. Incertain embodiments, the kit can include a set of printed instructionsor a label indicating that the kit is useful for the detection ofBacteroides, Porphyromonas, or Tannerella spp. In another embodiment,the kit provides reagents for the detection of Bacteroides,Porphyromonas, or Tannerella spp. nucleic acids. In one embodiment, thekit provides reagents for the PCR detection of Bacteroides,Porphyromonas, or Tannerella spp. nucleic acids and comprises in atleast one container a first isolated DNA molecule comprising a fragmentof at least about 15, 20, 25 or 30 nucleotides, which fragmenthybridizes under stringent conditions to a DNA molecule comprising asequence of at least 15, 30, 45, 60, 75, or 90 contiguous nucleotides,of any of the polynucleotides of SEQ ID NO:3-5, and a second isolatedDNA molecule comprising a fragment of at least 15, 20,25, or 30nucleotides, which fragment hybridizes under stringent conditions to aDNA molecule complementary to a DNA molecule having a sequence of atleast 15, 30, 45, 60, 75, or 90 contiguous nucleotides of any of thepolynucleotides of SEQ ID NO:3-5, which first and second DNA moleculescan be used to specifically amplify a Bacteroides, Porphyromonas, orTannerella spp. nucleic acid encoding a 16S rRNA which 16S rRNA isencoded by a DNA molecule selected from the group consisting of SEQ IDNOS: 3-5.

Vaccine Formulation and Method of Administration

The vaccine of the present invention can be administered to a companionanimal in an effective amount such that the vaccine therapeuticallytreats or confers resistance to or prevents periodontal disease in thecompanion animal. The vaccine of the present invention is useful in thecontrol of bacteria that cause periodontal disease. The vaccines of thepresent invention can, in particular, be used in the field of veterinarymedicine to treat companion animals and for the maintenance of publichealth against those bacteria described herein which are known to causeperiodontal disease.

The vaccines of the present invention are of value In the control ofbacteria that are injurious to, or spread or act as vectors of diseasein man and companion animals, for example those described herein. Thevaccines of the present invention are particularly useful in controllingbacteria that are present in companion animals for which purpose theycan be administered using any known methods of administration,including, but not limited to, oral, parenteral, intranasal,subcutaneous, or topical.

According to a further aspect of the present invention, there isprovided a composition comprising a vaccine of the present invention, inadmixture with a compatible adjuvant, diluent or carrier. In a preferredembodiment, the vaccine formulation of the present invention is composedof an aqueous suspension or solution containing at least one bacteria ofthe present invention and/or at least one subunit protein, preferablybuffered at physiological pH, in a form ready for injection.

The present invention further provides a method of treating orpreventing a bacterial infection, which comprises treatment with aneffective amount of a vaccine or vaccine formulation of the presentinvention. It is to be appreciated that reference to treatment includesprophylaxis as well as the alleviation of established symptoms of abacterial infection.

The vaccines and vaccine formulations of the present Invention can beused to induce a response that prevents the pathological changescharacteristic of periodontal disease caused by periodontaldisease-causing bacteria. In a vaccine formulation, an immunogenicamount of the bacteria, purified protein, nucleic acid, or combinationsthereof is desirably mixed with a suitable conventional vaccineadjuvants and physiologic vehicles, for use in mammals.

A vaccine formulation for preventing periodontal disease in companionanimals can be produced using at least one of the isolated and purifiedinactivated or attenuated bacteria, purified polypeptides (such asnative proteins, subunit proteins, or polypeptides) and admixing one ormore or these with a compatible adjuvant, diluent, or carrier.

The present invention further provides for combination vaccines havingat least one of the inactivated or attenuated bacteria in combinationwith one or more additional immunogenic components. Such a combinationvaccine produces in the vaccinated animal a surprisingly greater effectthan that expected by simply adding the effects of each componentadministered separately. Thus, a combination vaccine may stimulate asynergistic production of antibody in animals.

Other immunogenic components useful in the combination vaccines hereincontemplated include, but are not limited to, canine distemper (CD)virus, canine adenovirus type 2 (CAV-2), canine parainfluenza (CPI)virus, canine parvovirus (CPV), canine coronavirus (CCV), canineherpesvirus, and rabies virus. Antigens from these immunogens for use inthe vaccine compositions of the present invention can be in the form ofa modified live viral preparation or an inactivated viral preparation.Methods of attenuating virulent strains of these viruses and methods ofmaking an inactivated viral preparation are known in the art and aredescribed in, e.g., U.S. Pat. Nos. 4,567,042 and 4,567,043.

In accordance with the present invention, the combination vaccinesgenerally include a veterinary-acceptable carrier. Aveterinary-acceptable carrier includes any and all solvents, dispersionmedia, coatings, adjuvants, stabilizing agents, diluents, preservatives,antibacterial and antifungal agents, isotonic agents, adsorptiondelaying agents, and the like. Diluents can include water, saline,dextrose, ethanol, glycerol, and the like. Isotonic agents can includesodium chloride, dextrose, mannitol, sorbitol, and lactose, amongothers. Stabilizers include albumin, among others.

One or more antigens from other pathogens, and the veterinary-acceptablecarrier can be combined in any convenient and practical manner to form acombination vaccine composition, e.g., by admixture, solution,suspension, emulsification, encapsulation, absorption and the like, andcan be made in formulations such as tablets, capsules, powder, syrup,suspensions that are suitable for injections, implantations,inhalations, ingestions or the like.

Vaccines of the present invention can be prepared by combination of atleast one of the inactivated or attenuated bacteria with apharmaceutically acceptable carrier and, preferably, an adjuvant.

Suitable preparations of the vaccines of the present invention includeinjectables, either liquid solutions or suspensions. Solid formssuitable for solution in, or suspension in, a liquid pharmaceuticallyacceptable carrier prior to injection may also be prepared. The vaccinepreparation may be emulsified. The active immunogenic component, ispreferably mixed with an adjuvant which is pharmaceutically acceptableand compatible with the active immunogenic component. Suitable adjuvantsinclude, but are not limited to: mineral gels, e.g., aluminum hydroxide;surface active substances such as lysolecithin; glycosides, e.g.,saponin derivatives such as Quil A or GPI-0100 (U.S. Pat. No.5,977,081); cationic surfactants such as DDA, pluronic polyols;polyanions; non-ionic block polymers, e.g., Pluronic F-127 (B.A.S.F.,USA); peptides; mineral oils, e.g. Montanide ISA-50 (Seppic, Paris,France), carbopol, Amphigen (Hydronics, Omaha, Nebr. USA), Alhydrogel(Superfos Biosector, Frederikssund, Denmark) oil emulsions, e.g. anemulsion of mineral oil such as BayolF/Arlacel A and water, or anemulsion of vegetable oil, water and an emulsifier such as lecithin;alum, cholesterol, rmLT, cytokines and combinations thereof. Theimmunogenic component may also be incorporated into liposomes, orconjugated to polysaccharides and/or other polymers for use in a vaccineformulation. Additional substances that can be included in a product foruse in the present methods include, but are not limited to one or morepreservatives such as disodium or tetrasodium salt ofethylenediaminetetracetic acid (EDTA), merthiolate, and the like.

The subject to which the vaccine is administered is preferably acompanion animal, most preferably, a dog or cat.

It is preferred that the vaccine of the invention, when in a vaccineformulation, be present in unit dosage form. For purposes of thisinvention, an immunogenic amount, when administered comprises about1×10⁴ to about 1×10¹³ inactivated bacterial cells. In a vaccineformulation containing multiple components, the same or lesserimmunogenic amounts can usefully be employed.

Appropriate therapeutically effective doses can be determined readily bythose of skill in the art based on the above immunogenic amounts, thecondition being treated and the physiological characteristics of theanimal. Accordingly, a vaccine preparation provides a dosage of asterile preparation of an immunogenic amount of the activeingredient(s), where the active ingredient is at least one bacteria. Inthe presence of additional active agents, these unit dosages can bereadily adjusted by those of skill in the art.

A desirable dosage regimen involves administration of at least one doseof desired vaccine composition, where the antigenic content of eachfraction is as stated above. Effective doses (immunizing amounts) of thevaccines of the invention may also be extrapolated from dose-responsecurves derived from model test systems. The mode of administration ofthe vaccines of the invention can be any suitable route that deliversthe vaccine to the host. These include but are not limited to oral,intradermal, intramuscular, intraperitoneal, subcutaneous, intranasalroutes, and via scarification (scratching through the top layers ofskin, e.g., using a bifurcated needle). However, the vaccine ispreferably administered subcutaneously or by intramuscular injection.Other modes of administration can also be employed, where desired, suchas intradermally, intravenously, intranasally, or intratonsillarly.

Studies have shown that, for each of the above described vaccinecompositions, a primary immunization of young animals (after 8 weeks ofage) is desirably initiated, with booster doses administered at 12 weeksand 16 weeks of age. Annual re-vaccination is recommended.

The vaccine of the present invention is administered and dosed inaccordance with good medical practice, taking into account the clinicalcondition of the individual subject, the site and method ofadministration, scheduling of administration, subject age, sex, bodyweight and other factors known to medical practitioners.

The invention further provides kits for the prevention periodontaldisease in companion animals. In one embodiment, the kit provides acontainer comprising a therapeutically effective amount of acomposition, which prevents periodontal disease in companion animals.Also provided in the same or different container is a pharmaceuticallyacceptable carrier that may be used in the composition. The kit canadditionally include an adjuvant that can be used to aid in creating theresponse to the composition of the present invention. Also, the kit caninclude a dispenser for dispensing the composition, preferably in unitdosage form. The dispenser can, for example, comprise metal or plasticfoil, such as a blister pack. The kit can be accompanied by a label orprinted instructions describing administration of the composition toprevent periodontal disease in a companion animal. Compositionscomprising a vaccine composition of the present Invention formulated ina pharmaceutically acceptable carrier can also be prepared, placed in anappropriate container, and labeled for treatment of the indicatedperiodontal condition.

Determination of Vaccine Efficacy

The specific mechanism of protection induced by the vaccines and vaccinecompositions compositions of the present invention is the induction ofthe antibody and/or cellular immune response in vaccinated animals, asindicated by the in vivo animal tests described below.

The bacteria, vaccines, and vaccine compositions of the presentinvention are useful in treating or preventing companion animalperiodontal disease, bovine foot rot, coronary heart disease (dogs), orsystemic infections (dogs). The present invention is further illustratedby the following non-limiting example and accompanying tables.

EXAMPLE 1 Companion Animal Crevicular Fluid Sample

Microbial samples were taken from dogs and cats examined at veterinaryclinics for periodontal treatment, or dogs examined at certainrecognized facilities for normal check-ups. Dogs with periodontalpockets >3 mm and cats with periodontal pockets >2 mm were included inthis study. Dental indices (gingival index and periodontal index) andthe periodontal pocket depths were recorded. Individual coarse absorbentpaper points (Henry Schein; Melville, N.Y.) were aseptically insertedinto the periodontal pocket. Upon removal, the paper points wereimmediately inserted into vials containing Pre-Reduced AnaerobicallySterile (PRAS) Anaerobic Dental Transport (ADT) Medium (AnaerobeSystems; Morgan Hills, Calif.).

Vials were transferred into a Bactron IV anaerobic chamber (SheldonManufacturing, Cornelius, Oreg.) and processed under 90% N₂, 5% H₂, 5%CO₂. The paper points were aseptically placed into 50 μl of PRAS BrainHeart Infusion (BHI), PYG or SSYG media (Anaerobe Systems) and vortexedfor 30 seconds. Dilutions of 1:100 and 1:1000 were prepared in BHI, PYGor SSYG media. Aliquots of 100 μl of the 1:100 and 1:1000 dilutions werespread on PRAS Burcella Blood Agar (BRU) plates (Anaerobe Systems). Theplates were incubated at 37° C. in the anaerobic chamber for five toseven days. The total number of bacterial colonies and the number ofBlack Pigmented Anaerobic Bacteria (BPAB) colonies were counted.Individual BPAP colonies were transferred to new BRU plates andre-incubated as above.

Clinical Isolate Characterization

Each clinical isolate was subjected to a number of biochemical analysesand 16S rRNA DNA sequence analysis, using primers D0056 and D0057 (Seq.ID No. 1 and Seq. ID No. 2; Table 1), to determine genus and species.Individual isolates were streaked on BRU plates. Kanamycin, Vancomycin,and Colistin disks (Anaerobe Systems) were placed on the agar surface todetermine the KVC resistance patterns of each isolate. Purified colonieswere also subjected to the indole and catalase tests (Anaerobe Systems).Individual isolates were transferred to Egg Yolk Agar (EYA) plates(Anaerobe Systems) in order to determine lipase and lecithinaseproduction patterns. This data is shown in FIG. 1 below.

Periodontal Index refers to a systematic classification of the severityof periodontal disease, taking into account multiple aspects of thismultifaceted disease. These include, but are not limited to: pocketdepth, attachment loss, bleeding on probing, dental mobility, andgingivitis. Gingival Index refers to a systematic classification of theseverity of gingival inflammation. Signs observed which impact thisclassification include, but are not limited to: degree of edema, color,spontaneous bleeding, gingival recession, and hyperplasticity.

The partial 16S rRNA sequences from the five Tannerella forsythensisisolates characterized revealed 100% identity within the approximately520-bp region. The three Porphyromonas levii isolates were greater than99% identical within the partial 16S rRNA sequences analyzed, differingonly at one nucleotide (position 13 in SEQ ID NO. 4).

Identification of a Novel Species of Bacteroides (Bacteroidesdenticanoris)

During the course of this study we identified numerous clinical isolateswhose 16S rRNA sequences did not have highly similar matches in theavailable databases, indicating that the bacteria may represent novelisolates. One group of these isolates (Table 2-below) appeared torepresent a novel species. Based on the data presented herewithin, wepropose that this group of bacterial isolates be called Bacteroidesdenticanoris sp. nov. The type strain of B. denticanoris is strainB78^(T) (=ATCC PTA-5881). TABLE 2 Canine clinical bacterial isolatesutilized in this study. SEQ ID Periodontal NO. of 16S pocket RNA StrainTooth depth (mm) Location Sequence B denticanoris B78^(T) Upper left premolar #4 5 Pennsylvania 3 (fragment and 6 (full length) B. denticanorisB80 Upper left pre molar #4 5 Pennsylvania  7 B. denticanoris B83 Upperleft pre molar #4 5 Pennsylvania  8 B. denticanoris B241 Upper left premolar #4 4 Indiana  9 B. denticanoris B242 Upper left pre molar #4 4Indiana 10 B. denticanoris B342 Lower left first molar 5 Pennsylvania 11B. denticanoris B458 Upper right canine ND* California 12 B.denticanoris B473 Upper right pre molar #4 3 California 13 B.denticanoris B474 Upper right pre molar #4 3 California 14 B.denticanoris B476 Upper right pre molar #4 3 California 15*ND, not determinedPhenotypic Characterization of Bacteroides denticanoris B78^(T)

B. denticanoris B78^(T) was isolated from a five-year old female mixedbreed dog with periodontal disease. Clinically, the dog had aperiodontal index score of 3, and a gingival index score of 2 (Harvey,1998). A paper point sample was obtained from the upper left, fourthpremolar, which had a periodontal pocket depth of 5 mm. The sample wasprocess as described above. The purified cells were Gram-negative,non-spore forming, non-motile, rod shaped, and catalase-negative.Colonies formed after approximately five days of anaerobic incubation onBrucella blood agar at 37° C. Colonies of B. denticanoris B78^(T) beganto pigment (tan to black) after 5-7 days of Incubation. The isolateappeared hemolytic on Brucella blood agar, sensitive to kanamycin, andresistant to both vancomycin and colistin (antibiotic discs fromAnaerobe Systems). Colonies on egg yolk agar (Anaerobe Systems)demonstrated lecithinase activity, but not lipase activity. There was noevidence of bacterial swarming since distinct colonies appeared onnumerous media types (data not shown).

Biochemical Analysis

B. denticanoris B78^(T) was subjected to biochemical analysis using theRapID ANA II clinical test kit (Remel; Lenexa, Kans.). Briefly, threecultures of B. denticanoris B78^(T) on Brucella blood agar wereresuspended to McFarland # 3 equivalency. The suspensions were added tothe test wells and incubated for 4 hours at 37° C. Following theincubation, results for the 18 different biochemical tests wererecorded.

FIG. 2 shows the results of RapID ANA II testing for B. denticanorisB78^(T) as well as six control bacteria. Of the 18 tests performed bythe RapID ANA II kit, six (ONPG, βGLU, αFUC, NAG, PO₄, and LGY) werepositive for B. denticanoris B78^(T). In comparison, Porphyromonasgingivalis ATCC 33277, Prevotella intermedia ATCC 25611, Tannerellaforsythensis ATCC 43037, Bacteroides thetaiotaomicron ATCC 29148,Bacteroides fragilis ATCC 25285, and Bacteroides splanchnicus ATCC 29572yielded 5, 4, 10, 12, 9, and 8 positive tests, respectively.

Phylogenetic Analysis

The full-length 16S rRNA genes from B. denticanoris B78^(T) was PCRamplified in triplicate using the primers D134(5′-GAGTTTGATCCTGGCTCAGG-3′-SEQ ID NO:16) and D57(5′-CCCGGGAACGTATTCACCG-3′-SEQ ID NO:17) (Invitrogen Corp.). Slots, J.,et al. Clin Infect Dis 20 Suppl 2, S304-S307 (1995).

The PCR products were pooled, purified, desalted, and subjected todirect DNA sequence analysis. BLAST-N (Altschul, S. F. et al. J Mol Biol215, 403-410) (1990). searches of the non-redundant nucleotide databaseat the National Center for Biotechnology Information using the B.denticanoris B78^(T) 16S rRNA gene sequence indicated that the B.denticanoris B78^(T) isolate was related to members of the Bacteroidesgenus. The most closely related sequence in the database was the 16SrRNA gene sequence of Bacteroidetes sp. 0103 800 (accession numberAJ416906), showing 97% identity over 1,463 bp. Bacteroides sp. 0103-800was isolated from an anaerobic brain abscess.

Phylogenetic analysis based on 16S rRNA gene sequences was performedusing the CLUSTAL X version 1.81 software. Phylogenetic trees weregenerated using the neighbor-joining method (Saitou, N. & Nei, M. MolBiol Evol 4, 406-425) (1987).

Bootstrap values were obtained using 1000 replicates. FIG. 3 shows theresults of phylogenetic analysis for the B. denticanoris B78^(T)isolate. The placement of the major genera (Porphyromonas, Bacteroides,Prevotella, Tannerella, etc.) is in agreement with previously publishedphylogenetic trees for the cytophaga-flavobacter-bacteroides (CFB) group(Paster, et al (1994). J Bacteriol 176, 725-732., Shah, H. N., et al.Bacteroides, Prevotella, and Porphyromonas. In Microbiology andmicrobial infections, pp. 1305-1330. Edited by A. Balows and B. I.Duerden. London: Oxford University Press (1998).

B. denticanoris B78^(T) , Bacteroides sp. 0103-800, and the unculturedBacteroidetes Bisii27 isolate are grouped In an off branch of the B.fragilis group that also contains B. acidofaciens and B.thetaiotaomicron (FIG. 1). A bootstrap confidence value of 99.9% on thebranch point of the B. denticanoris B78^(T) sub-group from the B.fragilis sub-group adds strength to the phylogenetic placement of thisnewly identified organism.

An approximately 560-bp region of the 16S rRNA gene from nine othercanine clinical isolates of B. denticanoris (Table 1) was PCR amplified(in triplicate) using the D56 and D57 primers described above. The PCRproducts were purified, desalted, and pooled. The DNA sequence of thePCR products was then determined. The results are detailed below inTable 3. The isolates were typed based on their 16S rRNA DNA sequence.Individual, well-isolated colonies were utilized as template forpolymerase chain reactions (PCR) amplification of the 16S rRNA regionusing primers D0056 and D0057 (Seq. ID No. 1 and Seq. ID No. 2; Table 1)in triplicate. The PCR was carried out in 50 μl reaction volumescontaining 1× PCR buffer (Life Technologies; Rockville, Md.), 1.0 mMMgCl₂, 1.25 μM each primer, 300 μM each deoxy-NTP, and 2.5 U PlatinumPfx DNA Polymerase (Life Technologies). The following PCR cycleconditions were utilized: a two minute denaturation step at 94° C.; 30cycles of denaturation at 94° C. for 40 seconds, annealing at 60° C. for40 seconds, and extension at 72° C. for one minute; a final extensionstep at 72° C. for two minutes; and a final cooling step to 4° C. AGeneAmp 9700 thermocycler (Perkin Elmer Applied Biosystems; Foster City,Calif.) was utilized for all POR amplifications.

The resulting PCR products were purified using the PCR preps kits(Promega Corp.; Madison, Wisc.) and pooled by isolate. The purified PCRproducts were then desalted by drop analysis against 25 ml sterile waterusing a 0.025 μm nitrocellulose filter (Millipore Corp.; Bedford,Mass.). The purified, desalted PCR products were subjected to DNAsequence analysis using the DyeDeoxy termination reaction on an ABIautomated DNA sequencer. Synthetic oligonucleotide primers D0056 andD0057 (Seq. ID No. 1-2, respectively; 1) were used to obtain doublestranded DNA sequence. The resulting DNA sequences were used to searchpublicly available DNA databases using a BLAST-N program publiclyavailable from The National Center for Biotechnology Information, USA.TABLE 3 DNA sequence identification listing. All oligonucleotide primerswere synthesized by Gibco-BRL (USA). SEQ ID NO. Name DNA Sequence  7Bacteroides CAGTAAACGATGAATACTCGCTGTTTGCGATACACTGTAAGCGGCCAAGCGAAAdenticanoris GCGTTAAGTATTCCACCTGGGGAGTACGCCGGCAACGGTGAAACTCAAAGAA (B80)16S rRNA TTGACGGGGGCCCGCACAAGCGGAGGAACATGTGGTTTAATTCGATGATACGCpolynucleotide GAGGAACCTTACCCGGGCTTAAATTGCGCTGGCTTTTACCGGAAACGGTATTTTsequence CTTCGGACCAGCGTGAAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAAGTGCCATAACGAGCGCAACCCTTATCTTTAGTTACTAACAGTTTTGCTGAGGACTCTAAAGAGACTGCCGTCGTAAGATGCGAGGAAGGTGGGGATGACGTCAAATCAGCACGGCCCTTACGTCCGGGGCTACACACGTGTTACAATGGGGAGCACAGCAGGTTGCTACACGGCGACGTGATGCCAATCCGTAAAACTCCTCTCAGTTCGGATCGAAGTCTGCAACCCGACTTCGTGAAGCTGGATTCGCTAGTAATCGCGCATCAGCCACGGCGCGGTGAATAC  8 BacteroidesCAGTAAACGATGAATACTCGCTGTTTGCGATACACTGTAAGCGGCCAAGCGAAA denticanorisGCGTTAAGTATTCCACCTGGGGAGTACGCCGGCAACGGTGAAACTCAAAGGAA (B83) 16S rRNATTGACGGGGGCCCGCACAAGCGGAGGAACATGTGGTTTAATTCGATGATACGC polynucleotideGAGGAACCTTACCCGGGCTTAAATTGCGCTGGCTTTTACCGGAAACGGTATTTT sequenceCTTCGGACCAGCGTGAAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAAGTGCCATAACGAGCGCAACCCTTATCTTTAGTTACTAACAGTTTTGCTGAGGACTCTAAAGAGACTGCCGTCGTAAGATGCGAGGAAGGTGGGGATGACGTCAAATCAGCACGGCCCTTACGTCCGGGGCTACACACGTGTTACAATGGGGAGCACAGCAGGTTGCTACACGGCGACGTGATGCCAATCCGTAAAACTCCTCTCAGTTCGGATCGAAGTCTGCAACCCGACTTCGTGAAGCTGGATTCGCTAGTAATCGCGCATCAGCCACGGCGCGGTGAATAC  9 BacteroidesGCACAGTAAACGATGAATACTCGCTGTTTGCGATACACTGTAAGCGGCCAAGC denticanorisGAAAGCGTTAAGTATTCCACCTGGGGAGTACGCCGGCAACGGTGAAACTCAAA (B241) 16S rRNAGGAATTGACGGGGGCCCGCACAAGCGGAGGAACATGTGGTTTAATTCGATGAT polynucleotideACGCGAGGAACCTTACCCGGGCTTAAATTGCGCTGGCTTTTACCGGAAACGGT sequenceATTTTCTTCGGACCAGCGTGAAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAAGTGCCATAACGAGCGCAACCCTTATCTTTAGTTACTAACAGTTTTGCTGAGGACTCTAAAGAGACTGCCGTCGTAAGATGCGAGGAAGGTGGGGATGACGTCAAATCAGCACGGCCCTTACGTCCGGGGCTACACACGTGTTACAATGGGGAGCACAGCAGGTTGCTACACGGCGACGTGATGCCAATCCGTAAAACTCCTCTCAGTTCGGATCGAAGTCTGCAACCCGACTTCGTGAAGCTGGATTCGCTAGTAATCGCGCATCAACCACGGCGCGGTGAATA 10 BacteroidesACAGTAAACGATGAAATACTCGCTGTTTGCGATACACTGTAAGCGGCC denticanorisAAGCGAAAGCGTTAAGTATTCCACCTGGGGAGTACGCCGGCAACGGTGAAACT (B242) 16S rRNACAAAGGAATTGACGGGGGCCCGCACAAGCGGAGGAACATGTGGTTTAATTCGA polynucleotideTGATACGCGAGGAACCTTACCCGGGCTTAAATTGCGCTGGCTTTTACCGGAAA sequenceCGGTATTTTCTTCGGACCAGCGTGAAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAAGTGCCATAACGAGCGCAACCCTTATCTTTAGTTACTAACAGTTTTGCTGAGGACTCTAAAGAGACTGCCGTCGTAAGATGCGAGGAAGGTGGGGATGACGTCAAATCAGCACGGCCCTTACGTCCGGGGCTACACACGTGTTACAATGGGGAGCACAGCAGGTTGCTACACGGCGACGTGATGCCAATCCGTAAAACTCCTCTCAGTTCGGATCGAAGTCTGCAACCCGACTTCGTGAAGCTGGATTCGCTAGTAATCGCGCATCAACCACGGCGCGGTGAATA 11 BacteroidesCGCACAGTAAACGATGAATACTCGCTGTTTGCGATACACTGTAAGCGGCCAAG denticanorisCGAAAGCGTTAAGTATTCCACCTGGGGAGTACGCCGGCAACGGTGAAACTCAA (B342) 16S rRNAAGGAATTGACGGGGGCCCGCACAAGCGGAGGAACATGTGGTTTATTCGATGA polynucleotideTACGCGAGGAACCTTACCCGGGCTTAAATTGCGCTGGCTTTTACCGGAAACGG sequenceTATTTTCTTCGGACCAGCGTGAAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAAGTGCCATAACGAGCGCAACCCTTATCTTTAGTTACTAACAGTTTTGCTGAGGACTCTAAAGAGACTGCCGTCGTAAGATGCGAGGAAGGTGGGGATGACGTCAAATCAGCACGGCCCTTACGTCCGGGGCTACACACGTGTTACAATGGGGAGCACAGCAGGTTGCTACACGGCGACGTGATGCCAATCCGTAAAACTCCTCTCAGTTCGGATCGAAGTCTGCAACCCGACTTCGTGAAGCTGGATTCGCTAGTAATCGCGCATNACCACGGNGCGGTGAATAC 12 BacteroidesGCACAGTAAACGATGAATACTCGCTGTTTGCGATACACTGTAAGCGGCCAAGC denticanorisGAAAGCGTTAAGTATTCCACCTGGGGAGTACGCCGGCAACGGTGAAACTCAAA (B458) 16S rRNAGGAATTGACGGGGGCCCGCACAAGCGGAGGAACATGTGGTTTAATTCGATGAT polynucleotideACGCGAGGAACCTTACCCGGGCTTAAATTGCGCTGGCTTTTACCGGAAACGGT sequenceATTTTCTTCGGACCAGCGTGAAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAAGTGCCATAACGAGCGCAACCCTTATCTTTAGTTACTAACAGTTTTGCTGAGGACTCTAAAGAGACTGCCGTCGTAAGATGCGAGGAAGGTGGGGATGACGTCAAATCAGCACGGCCCTTACGTCCGGGGCTACACACGTGTTACAATGGGGAGCACAGCAGGTTGCTACACGGCGACGTGATGCCAATCCGTAAAACTCCTCTCAGTTCGGATCGAAGTCTGCAACCCGACTTCGTGAAGCTGGATTCGCTAGTAATCGCGCATCAGCCACGGCGCGGTGAATA 13 BacteroidesCACAGTAAACGATGAATACTCGCTGTTTGCGATACACGGTAAGCGGCCAAGCG denticanorisAAAGCGTTAAGTATTCCACCTGGGGAGTACGCCGGCAACGGTGAAACTCAAAG (B473) 16S rRNAGAATTGACGGGGGCCCGAACAAGCGGAGGAACATGTGGTTTAATTCGATGATA polynucleotideCGCGAGGAACCTTACCCGGGCTTAAATTGCGCTGGCTTTTACCGGAAACGGTA sequenceTTTTCTTCGGACCAGCGTGAAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAAGTGCCATAACGAGCGCAACCCTTATCTTTAGTTACTAACAGTTTTGCTGAGGACTCTAAAGAGACTGCCGTCGTAAGATGCGAGGAAGGTGGGGATGACGTCAAATCAGCACGGCCCTTACGTCCGGGGCTACACACGTGTTACAATGGGGAGCACAGCAGGTTGCTACACGGCGACGTGATGCCAATCCGTAAAACTCCTCTCAGTTCGGATCGAAGTCTGCAACCCGACTTCGTGAAGCTGGATTCGCTAGTAATCGCGCATCAGCCACGGCGCGGTGAATAC 14 BacteroidesACAGTAAACGATGAATACTCGCTGTTTGCGATACACGGTAAGCGGCCAAGCGA denticanorisAAGCGTTAAGTATTCCACCTGGGGAGTACGCCGGCAACGGTGAAACTCAAAGG (B474) 16S rRNAAATTGACGGGGGCCCGCACAAGCGGAGGAACATGTGGTTTAATTCGATGATAC polynucleotideGCGAGGAACCTTACCCGGGCTTAAATTGCGCTGGCTTTTACCGGAAACGGTAT sequenceTTTCTTCGGACCAGCGTGAAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAAGTGCCATAACGAGCGCAACCCTTATCTTTAGTTACTAACAGTTTTGCTGAGGACTCTAAAGAGACTGCCGTCGTAAAGATGCGAGGAAGGTGGGGATGACGTCAAATCAGCACGGCCCTTACGTCCGGGGCTACACACGTGTTACAATGGGGAGCACAGCAGGTTGCTACACGGCGACGTGATGCCAATCCGTAAAACTCCTCTCAGTTCGGATCGAAGTCTGCAACCCGACTTCGTGAAGCTGGATTCGCTAGTAATCGCGCATCAGCCACGGCGCGGTGAATAC 15 BacteroidesCACAGTAAACGATGAATACTCGCTGTTTGCGATACACGGTAAGCGGCC denticanorisAAGCGAAAGCGTTAAGTATTCCACCTGGGGAGTACGCCGGCAACGGTGAAACT (B476) 16S rRNACAAAGGAATTGACGGGGGCCCGCACAAGCGGAGGAACATGTGGTTTAATTCGA polynucleotideTGATACGCGAGGAACCTTACCCGGGCTTAAATTGCGCTGGCTTTTACCGGAAA sequenceCGGTATTTTCTTCGGACCAGCGTGAAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAAGTGCCATAACGAGCGCAACCCTTATCTTTAGTTACTAACAGTTTTGCTGAGGACTCTAAAGAGACTGCCGTCGTAAGATGCGAGGAAGGTGGGGATGACGTCAAATCAGCACGGCCCTTACGTCCGGGGCTACACACGTGTTACAATGGGGAGCACAGCAGGTTGCTACACGGCGACGTGATGCCAATCCGTAAAACTCCTCTCAGTTCGGATCGAAGTCTGCAACCCGACTTCGTGAAGCTGGATTCGCTAGTAATCGCGCATCAGCCACGGCGCGGTGAATAC

The partial 16S rRNA sequences from the ten B. denticanoris isolateswere found to cluster into four sequences groups (B78^(T), B80, B83,B342, and B458; B241; B242; and B473, B474, and B476). All isolateswithin a group had identical 16S rRNA sequences in this approximately560-bp region. FIG. 4 shows the results of phylogenetic analysis of theB. denticanoris isolates partial 16S rRNA gene sequences. Between all ofthe B. denticanoris isolates, there is a 99.5% DNA sequence identitywithin the 560-bp region. Based on this observation, we conclude thatall of these isolates are varying strains of the same species.Additionally, strains of this species were found in geographicallydistant locations (Pennsylvania and California).

A complete listing of all the isolates and their respectivecharacteristics is located in FIG. 1.

The distribution of isolates is shown in Table 4. TABLE 4 Summary of thenumber of dogs identified to harbor indicated bacterial species. # dog #% positive Isolate isolates dogs dogs Bacteroides denticanoris 10 5 10Porphyromonas levii 3 2 4 Tannerella forsythensis 5 4 8

The table above indicates the number of isolates, as well as the numberand percentage of dogs from which the indicated bacterial species wereisolated.

The following companion animal periodontal isolates were deposited withthe American Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Va., 20110, USA, Bacteroides denticanoris (B78;(PTA-5881),Porphyromonas levii (B222; (PTA-5882), and Tannerella forsythensis(B343-24; PTA-6063).

Culture Conditions for Bacterial Species

Since the standard growth media for many anaerobic bacteria (Brain HeartInfusion [BHI] and Chopped Meat Carbohydrate [CMC] media) contain animalproduct, which are not amenable for vaccine production, a growth mediumthat does not contain these ingredients was sought. Various mediacompositions, with and without the addition of hemin and vitamin K, weretested for their ability to support growth equivalent to that of growthof BHI or CMC. Both the PYG-complete and the SSYG media supported thegrowth of Bacteroides denticanis, P. levii, and T. forsythensis. ThePYG-complete or SSYG-complete media were chosen as the growth media dueto their ability to yield high-density cultures during fermentation. ThePYG medium contains the following ingredients: 3% phytone (BectonDickinson; Cockeysville, Md.), 0.3% yeast extract (Becton Dickinson),0.3% glucose (Sigma Corp.; St. Louis, Mo.), 0.05% sodium thioglycollate(Becton Dickinson), 0.5% sodium chloride (Sigma Corp.), 5 μg/ml hemin(Sigma Corp.) (added after autoclaving), 0.5 μg/ml menadione (SigmaCorp.) (added after autoclaving), and 0.2% sodium bicarbonate (SigmaCorp.), pH 7.0. Bacteroides denticanis, P. levii, and T. forsythensis.were routinely cultivated on Brucella blood agar plates (AnaerobeSystems) or in complete PYG medium or BHI at 37° C. in a Bactron IVanaerobic chamber (Shel Labs; Cornelius, Oreg.) under 90% N₂, 5% CO₂ forthree to five days (plates) or 24 to 48 hours (liquid cultures). TheSSYG medium contains the following ingredients: 5% soytone (BectonDickinson), 0.3% yeast extract (Becton Dickinson), 0.3% glucose (EMIndustries), 0.05% sodium thioglycollate (Becton Dickinson), 0.5% sodiumchloride (Sigma Corp.), 0.2% sodium bicarbonate (Fisher), dH2O, pH 7.0and hemin-menadione solution containing hemin solution of 5 μg/ml hemin,1N sodium hydroxide in dH2O and menadione solution of 0.5 μg/ml in 95%ethanol.

Pathogenicity Testing of Clinical Isolates

Bacteroides denticanoris and P. levii were tested for theirpathogenicity in the mouse periodontal bone loss model. Three-week-old,age-matched male Balb/c CyJ mice (Jackson Laboratories; Bar Harbor, Me.)with estimated weights of 14-15 grams were utilized for this study. Theanimals were housed in positive pressure, barrier cage units. Foodpellets, standard for the species, and water were provided ad libitumthroughout the experiment. The bedding utilized was granular Bed O'Cobbsto minimize impaction in the gingival tissues. Following receipt, allanimals were acclimatized for five to seven days. To reduce competingoral flora, animals were placed on a mixture of sulfamathoxazole andtrimethoprim (10 ml drinking water; approximately 2 mg and 0.4 mg/ml,respectively) for ten days followed by a five-day washout period. Serumsamples were taken from each mouse tail vein bleed. The animals wereinfected with 0.5 ml suspension of approximately 1×10¹⁰ cfu/ml of theappropriate bacterial strain in 1% carboxymethylcellulose by gavage.Additional drops were placed in the oral cavity. This infection wasrepeated two more times for a total of three times (Monday, Wednesday,and Friday).

Day 1 of the experiment was defined as the Tuesday following the firstinfection. All animals were sacrificed on Day 2. Post-infection serumwas collected, as were microbial samples. The jaws of each mouse weredefleshed, stained, and scored for horizontal bone loss microscopically.The scoring was repeated three times to reduce operator error. Theaverage bone loss is expressed as the average bone loss/site/jaw in mm.Statistical analysis of the resulting data was done with Systat (version9), SigmaStat (version 2), and SigmaPlot (version 2000) available fromSPSS Science Inc. (Chicago, Ill.). Table 5 shows the numerical resultsfor these isolates. TABLE 5 Summary of the mouse periodontal diseasepathogenicity trial. Mean Bone Standard Isolate Mice/group Loss (mm)Deviation SEM Sham 16 3.35 0.473 0.383 Bacteroides 16 3.86 0.605 0.486denticanoris P. levii 16 3.53 0.460 0.371 T. forsythensis ND ND ND NDND = Not Done

These data indicate that the Bacteroides denticanoris isolate is capableof causing bone loss in the mouse model of periodontal disease. Althoughminimal, the Porphyromonas levii isolate did cause bone loss in themouse periodontal model. The results for Bacteroides denticanoris aredisplayed graphically in FIG. 5.

Throughout this application, various patent and scientific publications,including United States patents, are referenced by author and year andpatents by number. The disclosures of these publications and patents arehereby incorporated by reference in their entireties into thisapplication in order to more fully describe the state of the art towhich this invention pertains.

1. At least one isolated pigmented anaerobic bacterium comprising a 16SrRNA DNA sequence at least 95% homologous to a sequence selected fromthe group consisting of SEQ ID NOS: 3, 4, 5, 6, 9, 10 and 13 wherein thebacterium causes, either directly or in combination with otherpathogenic agents periodontal disease in companion animals.
 2. Thebacterium according to claim 1 comprising a 16S rRNA DNA sequence atleast 99% homologous to a sequence selected from the group consisting ofSEQ ID NOS: 3, 4, 5, 6, 9, 10 and
 13. 3. The bacterium according toclaim 1 comprising a 16S rRNA DNA sequence at least 99.5% homologous toa sequence selected from the group consisting of SEQ ID NOS: 3, 4, 5, 6,9, 10 and
 13. 4. The bacterium according to claim 1 comprising a 16SrRNA DNA sequence selected from the group consisting of SEQ ID NOS: 3,4, 5, 6, 9, 10 and
 13. 5. The bacterium according to claim 1 which isBacteroides denticanoris.
 6. The bacterium according to claim 5 which isATCC PTA-5881 or a bacterium having all of the identifyingcharacteristics of ATCC PTA-5881.
 7. The bacterium according to claim 1which is Porphyromonas levii.
 8. The bacterium according to claim 7which is ATCC PTA-5882 or a bacterium having all of the identifyingcharacteristics of ATCC PTA-5882
 9. The bacterium according to claim 1which is Tannerella forsythensis
 10. The bacterium according to claim 9which is ATCC PTA-6063 or a bacterium having all of the identifyingcharacteristics of ATCC PTA-6063
 11. The bacterium according to claim 1wherein the companion animal is a cat or a dog.
 12. An immunogeniccomposition comprising the pigmented anaerobic bacterium according toclaim
 1. 13. The immunogenic composition of claim 12 wherein thepigmented anaerobic bacterium is inactivated.
 14. The immunogeniccomposition of claim 12 further comprising a pharmaceutically acceptablecarrier.
 15. A vaccine for treating or preventing periodontal disease incompanion animals comprising an immunologically effective amount of thebacterium according to claim 1 and a pharmaceutically acceptablecarrier.
 16. The vaccine of claim 15 wherein the bacterium isinactivated.
 17. The vaccine composition as in claim 15, furthercomprising an adjuvant.
 18. A method for treating or preventingperiodontal disease in companion animals comprising administering to acompanion animal in need thereof, a vaccine composition according toclaim
 15. 19. A method for diagnosing periodontal disease in companionanimals by analyzing a sample from the oral cavity of the companionanimal wherein the presence of one or more pigmented anaerobic bacteriaaccording to claim 1 in the sample is indicative of disease.
 20. Themethod according to claim 19 wherein the presence of a polynucleotidecomprising a 16S rRNA DNA sequence at least about 95% homologous to asequence selected from the group consisting of SEQ ID NOS: 3, 4, 5, 6,9, 10 and 13 in the sample is indicative of disease.
 21. The methodaccording to claim 20 wherein the presence of a polynucleotidecomprising a 16S rRNA DNA sequence at least about 99% homologous to asequence selected from the group consisting of SEQ ID NOS: 3, 4, 5, 6,9, 10 and 13 in the sample is indicative of disease.
 22. The methodaccording to claim 20 wherein the presence of a polynucleotidecomprising a 16S rRNA DNA sequence at least about 99.5% homologous to asequence selected from the group consisting of SEQ ID NOS: 3, 4, 5, 6,9, 10 and 13 in the sample is indicative of disease.
 23. The methodaccording to claim 20 wherein the presence of a polynucleotidecomprising a 16S rRNA DNA sequence selected from the group consisting ofSEQ ID NOS: 3, 4, 5, 6, 9, 10 and 13 in the sample is indicative ofdisease.
 24. The method according to claim 19, wherein said analyzingstep includes analyzing the sample using a method selected from thegroup consisting of PCR, hybridization, and antibody detection.
 25. Akit comprising, in at least one container, a composition for treatingand preventing periodontal disease in companion animals comprising aneffective amount of at least one live or inactivated isolated pigmentedanaerobic bacteria, of any of claims 1 through 11 and a pharmaceuticallyacceptable carrier; wherein the kit further comprises a set of printedinstructions indicating that the kit is useful for treating orpreventing periodontal disease in companion animals.
 26. A kit accordingto claim 25, wherein said kit further comprises a means for dispensingsaid composition.
 27. A kit comprising in at least one container anisolated DNA molecule comprising a nucleotide sequence of at least about15 contiguous nucleotides selected from any of SEQ ID NOS: 3, 4, 5, 6,9, 10 and 13, which hybridizes under highly stringent conditions to thecomplement of any of the nucleotide sequences depicted in SEQ ID NOS: 3,4, 5, 6, 9, 10 and 13, and a second isolated DNA molecule comprising ina second container an isolated DNA molecule comprising a nucleotidesequence of at least about 15 contiguous nucleotides selected from thecomplement of any of the nucleotide sequences depicted in SEQ ID NOS: 3,4, 5, 6, 9, 10 and 13 which hybridizes under highly stringent conditionsto any of the nucleotide sequences depicted in SEQ ID NOS: 3, 4, 5, 6,9, 10 and 13, wherein the kit further comprises a set of instructionsindicating that the kit is useful for the detection of Bacteroides,Porphyromonas, or Tannerella spp.
 28. A hybridization kit comprising inat least one container an isolated DNA molecule comprising a nucleotidesequence of at least about 15 contiguous nucleotides selected from anyof SEQ ID NOS: 3, 4, 5, 6, 9, 10 and 13, or its complement, wherein thehybridization is specific to Bacteroides, Porphyromonas, or Tannerellaspp. and wherein the kit further comprises a set of instructionsindicating that the kit is useful for the detection of Bacteroides,Porphyromonas, or Tannerella spp.
 29. The kit according to claim 28wherein the hybridization is performed under highly stringentconditions.
 30. A biologically pure culture of bacteria wherein thebacteria comprise a 16S rRNA DNA sequence at least about 99% homologousto a sequence selected from the group consisting of SEQ ID NOS: 3, 6, 9,10 and
 13. 31. The biologically pure culture of bacteria according toclaim 30 wherein the 16S rRNA DNA sequence is at least about 99.5%homologous to a sequence selected from the group consisting of SEQ IDNOS: 3, 6, 9, 10 and
 13. 32. The biologically pure culture of bacteriaaccording to claim 30 wherein the 16S rRNA DNA sequence is selected fromthe group consisting of SEQ ID NOS: 3, 6, 9, 10 and
 13. 33. Thebiologically pure culture of bacteria according to claim 30, wherein thebiologically pure culture of bacteria is independently selected from:ATCC PTA-5881 or a culture having all of the identifying characteristicsof ATCC PTA-5881; ATCC PTA-5882 or a culture having all of theidentifying characteristics of ATCC PTA-5882: or ATCC PTA-6063 or aculture having all of the identifying characteristics of ATCC PTA-6063.34. A biologically pure culture of bacteria which is ATCC PTA-5882 or aculture having all of the identifying characteristics of ATCC PTA-5882.35. A biologically pure culture of bacteria which is ATCC PTA-6063 or aculture having all of the identifying characteristics of ATCC PTA-6063